tissue: LuCaP 35CR PDX tumor timepoint group: Day 7 treatment group: control
Treatment protocol
Animals were treated by oral gavage on a weekly schedule of 5 days on 2 days off, tumor volume and body weight were measured twice weekly, and blood samples were drawn weekly for prostate-specific antigen (PSA) measurements using AxSym Total PSA Assay (Abbott Laboratories, Abbott Park, IL). Five animals in each group were sacrificed 7 days after the initiation of treatment (D7) and the rest of the animals were followed and sacrificed until tumors exceeded 1000 mm3 (End of Study, EOS) or sacrificed if animals became compromised. At sacrifice (D7 or EOS), half of the tumor was harvested for paraffin embedding and half was frozen for intratumoral androgen measurement, DNA and RNA extraction.
Growth protocol
Animal procedures were carried out in accordance with NIH guidelines and upon University of Washington Institutional Animal Care and Use Committee approval. Four different LuCaP human prostate cancer PDXs (LuCaP 136CR, LuCaP 77CR, LuCaP 96CR, and LuCaP 35CR) were utilized. Intact male CB-17 SCID mice (~6 weeks of age, Charles River Laboratories, San Diego, CA) were implanted subcutaneously with tumor bits of LuCaP 136 or LuCaP 77. Mice were castrated when tumor volume was ≥100 mm3. When tumor regrew to 1.5-fold of the original volume, tumors were referred to as LuCaP 136CR or LuCaP 77CR and were randomized to vehicle (20% HPbCD/0.37N HCl/PBS) or AA treatment groups (0.5 mmol/kg, Janssen Pharmaceutica, Beerse, Belgium). LuCaP 96CR and LuCaP 35CR are castration-resistant PDXs that are propagated in castrated male mice. Castrated male CB-17 SCID mice were implanted subcutaneously with LuCaP 96CR or LuCaP 35CR tumor bits. When tumor volume ≥ 100 mm3, mice were randomized to vehicle or AA treatment groups (0.5 mmol/kg).
Extracted molecule
total RNA
Extraction protocol
Frozen pieces of tumor were attached to Optimal Cutting Temperature Compound and 5-µm sections were stained with H&E. Areas of viable tumor cells were identified and macro-dissected for RNA extraction using a standard procedure with RNA STAT 60 (Tel-Test, Friendswood, TX). RNA was then purified using RNeasy Mini kit utilizing the optional DNase digestion in solution prior to purification (Qiagen, Hilden, Germany) for subsequent gene expression analyses.
Label
biotin
Label protocol
Biotin-labeled, amplified RNA (aRNA) was synthesized from 200 ng total RNA using the 3′ IVT Express Kit (Affymetrix, Santa Clara, CA). The aRNA was purified using Agencourt RNAClean XP beads (Beckman Coulter Inc., Brea, CA) on the BioMek FX Workstation (Beckman Coulter Inc.). Biotin-labeled aRNA was fragmented using the 3′ IVT Express Kit.
Hybridization protocol
A total of 4.5 µg fragmented biotin-labeled aRNA was hybridized on an HT Human Genome (HG)-U219 96-array plate following the manufacturer's protocol.
Scan protocol
The plate was washed, stained, and scanned with the GeneTitan Instrument following the manufacturer's protocol.
Description
LuCaP 35CR_Day 7: control, sample: 7
Data processing
Gene expression microarray data were normalized to minimize systematic technical variation using robust multichip average (RMA) and represented in the log2 scale.
